Horseshoe crab amebocyte lysate factor G activation inhibitor

ABSTRACT

This invention relates to a horseshoe crab amebocyte lysate factor G activation inhibitor comprising as an active ingredient a polyglycoside containing at least one poly-(1→3)-β-D-glucoside structure portion consisting of 2 to 370 (1→3)-β-D-glucoside structural units of the following formula ##STR1## which are continuously bound to one another. This inhibitor is useful for inhibiting the activation of factor G which may exist in horseshoe crab amebocyte lysate used in the Limulus test.

This application is a continuation, of application Ser. No. 07/822,740,filed Jan. 21, 1992, now abandoned, which is a divisional application ofSer. No. 07/474,057, filed May 1, 1990, now U.S. Pat. No. 5,155,032,issued Oct. 13, 1992.

TECHNICAL FIELD

This invention relates to an agent which inhibits activation of acertain enzyme precursor, and more specifically, to a factor Gactivation inhibitor, or a substance which inhibits activation of a(1→3)-β-D-glucan sensitive factor, factor G, of a system whosecoagulation is started by reaction with (1→3)-β-D-glucan among factorsinvolved in the clotting system of horseshoe crab amebocyte lysate.

BACKGROUND TECHNOLOGY

In 1964, Levin and Bang discovered the phenomenon in which horseshoecrab (limulus) amebocyte lysate (hereinafter sometimes abbreviated asLAL) is immediately coagulated (gelled) by a Gram-negative bacterialendotoxin [J. Levin and F. B. Bang: Bull. Johns Hopkins Hospital, 115,265-274 (1964)]. Since then, LAL has been widely utilized as theso-called Limulus Test reagent in a method of specific detection ofendotoxins. Today, three genera, four species of horseshoe crab survivethroughout the world and Limulus polyphemus, Tachypleus tridentatus,Tachypleus gigas and Carcinoscorpius rotundicauda are known. "LimulusTest" reagents comprising the amebocyte lysate of L. Polyphemusoccurring in the United States and T. tridentatus occurring in Japan andChina have been commercialized. [See, for example, Progress in Clinicaland Biological Research: volume 93; "Endotoxins and their Detection withthe Limulus Amebocyte Lysate Test", edited by Stanly W. Watson, JackLevin and Thomas J. Novitsky, published in 1982 by Alan R. Lisps Inc.,pages 7-24, entitled: The Limulus Test and Bacterial Endotoxins: SomePerspectives by J. Levin.]

LAL was first considered to react specifically only with endotoxins.Recent studies have shown that LAL has been found to react with(1→3)-β-D-glucan as well as endotoxin. The coagulation system of LAL,like the mammalian blood coagulation system, consists of two or morecascade reactions of coagulation factors, and include not only anendotoxin-mediated pathway (factor C pathway), but also a pathway to betriggered by (1→3)-β-D-glucan (factor G pathway) [T. Morita et al., FEBSLETTERS, 129, 318-321 (1981), and S. Iwanaga et al., J. Protein Chem.,5, 255-268 (1986)]. Accordingly, work has been done in order to renderthe Limulus test as endotoxin-specific as possible. For example, T.Obayashi et al., Clin. Chim. Acta, 149, 55-65 (1985) proposed a methodof determining an endotoxin by using a reagent obtained by removingfactor G from LAL by separation and reconstitution of the coagulationfactors. An endotoxin specific assay kit in accordance with this methodis sold under the tradename "Endospecy®" by Seikagaku Kogyo Co., Ltd.

The above-proposed assay method has a very strong demand as anendotoxin-specific assay. However, this method involves certaindisadvantages to be described.

(1) To separate and remove factor G from limulus amebocyte lysatecomposed of a plurality of coagulation factors, it is necessary toperform the operation of separating the individual factors in theabsence of an endotoxin or (1→3)-β-D-glucan. Accordingly, the endotoxinor (1→3)-β-D-glucan must be removed completely in advance from tools,devices and chemicals used in the separating operation forfractionation.

(2) As the separating operation proceeds, the amebocyte lysate becomesdiluted, and it must occasionally be concentrated.

(3) Every time a separating operation is carried out, the factorsdecrease in activity or a loss of the fractions occurs.

(4) The coagulogen (clottable protein precursor) is separated andremoved together with factor G.

Because of these disadvantages, the above proposed assay method can beapplied only to a chromogenic method, and cannot be applied to methodsutilizing the gellation phenomenon such as a gellation method,turbidimetry and turbidimetric kinetic assay. In the course of studyinga pathway to be triggered by (1→3)-β-D-glucan (factor G pathway) in theLAL coagulation mechanism, the present inventors unexpectedly found thatamong (1→3)-β-D-glucans heretofore considered to be involved only in theactivation of factor G, one containing a structural portion consistingof a specific number of continuously bound (1→3)-β-D-glucosidestructural units, quite contrary, shows a factor G inhibiting action.This finding has led to accomplishment of the present invention.

DISCLOSURE OF THE INVENTION

The present invention provides a horseshoe crab amebocyte lysate factorG activation inhibitor comprising as an active ingredient apolyglycoside containing at least one poly-(1→3)-β-D-glucoside structureportion consisting of 2 to 370 (1→3)-β-D-glucoside structural units(molecular weight 162) of the following formula ##STR2## which arecontinuously bound to one another.

The present invention also provides a method of inhibiting theactivation of factor G existing in limulus amebocyte lysate (LAL), whichcomprises adding an effective amount of the polyglycoside to the limulusamebocyte lysate.

The present invention will be explained in more detail.

The polyglycoside used as an active ingredient in the inhibitor of thisinvention contains per molecule at least one poly-(1→3)-β-D-glucosidestructure portion consisting of 2 to 370, preferably 3 to 310, morepreferably 4 to 180, (1→3)-β-D-glucoside structural units of thefollowing formula ##STR3## continuously bound to one another [thispoly-(1→3)-β-D-glucoside structure portion is referred to simply as the"poly-(1→3)glucoside structure portion"].

So long as the polyglycoside used in this invention contains at leastone poly-(1→3)glucoside structure portion per molecule, the structure ofthe remaining portion of the polyglycoside molecule is not particularlylimited, and may be selected widely. It is important however that theother structural portion should not substantially react with endotoxinsand the factor C activation system. For example, the polyglycoside usedin this invention consists substantially of one poly-(1→3)glucosidestructure portion, for example poly-(1→3)-β-D-glucoside represented bythe following formula ##STR4## wherein n is an integer of 2 to 370,preferably 3 to 310, more preferably 4 to 180.

Alternatively, it may be of a structure resulting from binding of acarbohydrate chain composed of at least one (1→4)-β-D-glucosidestructural unit represented by the following formula ##STR5## and/or atleast one (1→6)-β-D-glucoside structural unit represented by thefollowing formula ##STR6## and/or at least one modified β-D-glucosidestructure of the formula ##STR7##

In the above formulae (IV), (V) and (VI), at least one of R₁, R₂ and R₃represents a chemically introducible functional group, such as a methylgroup, a hydroxymethyl group, a carboxymethyl group, an acetyl group, asulfuric acid group or a phosphoric acid group, or a metal salt, anammonium salt or an organic amine salt thereof, and the remainderrepresents a hydrogen atom.

The above carbohydrate chain may be bound as a branched chain to thepoly-(1→3)glucoside structure portion).

Furthermore, the polyglycoside used in this invention may be one inwhich two or more poly-(1→3)glucoside structural portions are linkedwith other carbohydrate chain structural portions interposed between thepoly-(1→3)glucoside structural portions as shown by the followingformula

    A.sub.1 -B.sub.1 -A.sub.2 -B.sub.2 - - -

wherein each of A₁, A₂ . . . represents a poly-(1→3)-β-D-glucosidestructure portion composed of 2 to 370, preferably 3 to 310, morepreferably 4 to 180 (1→3)-β-D-glucoside structural units of formula (I)continuously bound to one another, the number of the units of formula(I) in the structural portions A₁, A₂, . . . may be different, and B₁,B₂, . . . represent other carbohydrate chain structural portions whichare identical or different.

The other carbohydrate chain structural portions represented by B₁, B₂,. . . may be, for example, structural portions composed of onestructural unit of formula (II), (III), (IV), (V) or (VI) or two or moresuch structural units.

Furthermore, the polyglycoside used in this invention may be of such astructure that the above poly-(1→3)glucoside structural portion iscomposed of long-chain poly-(1→3)-β-D-glucoside structural portions eachof which is composed of at least 371 (1→3)-β-D-glucoside structuralunits of formula (I) continuously bound to one another, with the othercarbohydrate chain structures of formula B₁, B₂ . . . interrupting thechain.

Accordingly, the polyglycoside used in this invention is notparticularly limited in molecular weight so long as it contains at leastone poly-(1→3)glucoside structural portion per molecule. Conveniently,however, it generally has a molecular weight of not more than 500,000,preferably 500 to 240,000, more preferably 650 to 80,000. If itsmolecular weight falls outside the above range, its solubility in wateris reduced, and the viscosity of the solution increases to make itdifficult to handle, or disadvantages will arise in preparing aendotoxin assay kit of a consistent quality.

Preferably, the polyglycoside used in this invention consistssubstantially of at least one poly-(1→3)glucoside structural portion permolecule. It may, however, contain another polyglycoside containing ahigh-molecular-weight poly-(1→3)-β-D-glucoside structural portioncomposed of at least 371 (1→3)-β-D-glucoside structural units of formula(I) continuously bound to each other. This is because the polyglycosidein accordance with this invention binds to initiation factor G whichinitiates factor G activation of the factor G pathway of LAL morerapidly and strongly than the high-molecular-weightpoly-(1→3)-β-D-glucoside which is a factor G activating substance andthus inhibits activation of factor G and its inhibiting action is notsubstantially affected by the presence of the high-molecular-weightpoly-(1→3)-β-D-glucoside.

When the polyglycoside containing another component is used as aninhibitor, the amount of the polyglycoside in the inhibitor is notparticularly limited. However, if its amount is too small, thepolyglycoside must be used in a large amount to inhibit factor G, andthis is not economical. Generally, the amount of the polyglycoside inthe inhibitor is desirably at least 5% by weight, preferably at least10% by weight, more preferably at least 20% by weight.

The molecular weight of the polyglycoside of the present invention isdetermined by performing gel permeation chromatography under theconditions indicated below using a standard substance of a knownmolecular weight, drawing a standard curve, then performing gelpermeation chromatography under the same conditions on a test sample,and comparing the results with the standard curve.

Conditions used in gel permeation chromatography

Column: TSK gel G-PWXL series (Tosoh Co., Ltd.) 7.8×300 mm, severalcolumns of several types

Mobile phase: 0.3M NaOH

Flow rate: 0.5 ml/min.

Sample concentration: 0.1-5 mg/ml

Sample volume injected: 0.1 ml

Column temperature: room temperature

Method of detection: Measurement by a differential refractometer (madeby LKB Company), or carbohydrate assay by the phenol-sulfuric acidmethod.

Standard substance: TSK standard polyethylene oxide (a product of TosohCo., Ltd.) and polyethylene glycol (a product of Nakarai Chemicals Co.,Ltd.), 10 types having a weight average molecular weight ranging from1,000 to 860,000.

The polyglycoside having the above-described properties used as a factorG activation inhibitor in this invention may be one derived from nature,or a synthetic product, or a partially chemically modified product of apoly-(1→3)-β-D-glucoside containing at least three (1→3)-β-D-glucosidestructural units of formula (I). Usually, polyglycosides derived fromnature are easy to obtain. Specific examples of the polyglycoside aregiven below.

(1) Substantially straight-chain polyglycosides composed substantiallyof the (1→3)-β-D-glucoside structural units of formula (I) alone, forexample (1→3)-β-D-glucans derived from bacteria of the genusAlcaligenes; paramylon derived from flagellates (Euglena); β-glucansfrom the fibrous tissues of higher plants or callose extracted from thesieve tube; D-glucose polymer composed of (1→3)-β-bonds of higherdegrees of polymerization contained in partial hydrolyzates oflaminarans derived from brown algae of the genus Laminaria or Eisenia,or (1→3)-β-D-glucans; laminaridextrins having degrees of polymerizationof 10 to 20; and laminarioligosaccharides having degrees ofpolymerization of less than 10.

(2) Polyglycosides containing both the poly-(1→3)-β-D-glucosidestructural units of formula (I) and the (1→6)-β-D-glucoside structuralunits of formula (III). Examples ace given below.

(a) Polyglycosides in which glucose or a glucose polymer resulting fromlinking of one to several (1→6)-β-bonds with a main chain composed of(1→3)-β-bonds is incorporated, such as laminarans derived from brownalgae of the genus Eisenia.

(b) Polyglycosides resulting from attaching a carbohydrate chain of(1→3)-β-bonds through (1→6)-β-bond to the glucose or glucose polymerlinked by (1→3)-β-bonds in (a), which may further include anothercarbohydrate portion in a part of the carbohydrate chain, for examplelaminatans derived from brown algae of the genus Laminaria,chrysolaminarans derived from diatoms such as Ochromonas, Phaeodactylum,Skeletonema, Biddulphia, Coscinodiescus and Chaetoceros and pachymanderived from Poria.

(c) Polyglycosides having more branches and being dendriform, such as aglucan derived from the cell wall of Phytophthora.

(d) polyglycosides in which glucose is linked through (1→6)-β-bonds withstraight-chain glucan composed of (1→3)-β-bonds, such as sclerotanderived from Sclerotinia having a single glucosyl branch on every 3glucosyl residues of the main chain, schizophyllan from Schizophyllum,scleroglucans derived from Sclerotium, Corticium and Stromatinia, andpolyglycosides in which glucose is bound to a straight-chain glucancomposed of (1→3)-β-bonds via (1→6)-β-bonds at a rate of two glucosylresidues per 5 glucose units of the straight-chain glucan, such aslentinan of Lentinus.

(e) A straight-chain glucan composed of (1→6)-β-bonds having a pluralityof (1→3)-β-glucose chains branched from the C-3 portion of glucoseresidue of the main chain, such as β-glucan derived from the cell wallof Saccharomyces (bakers' yeast).

(3) Polyglycosides containing both the (1→3)-β-D-glucoside structuralunits of formula (I) and the (1→4)-β-D-glucoside structural units offormula (II), for example lichenans derived from Cetraria, Usnea andEvernia, and β-glucan contained in barley albumin, which are composed ofthe polyglycoside consisting of portions of (1→4)-β-oligoglucoside islinked through (1→3)-β-bonds containing (1→3)-β-oligoglycosides atintervals.

Some of the polyglycosides described above are commercially available,and may be utilized directly as the inhibitor of this invention. Asrequired, the carbohydrate chain is partially decomposed and/orsubjected to a separation treatment, and a fraction rich in apolyglycoside comprising the aforesaid specific amount of the(1→3)-β-D-glucoside structural units of formula (I) is prepared. Thisfraction may be utilized as the inhibitor of the invention.

The partial decomposition and the separation treatment of thecarbohydrate chain may be carried out by known methods. The partialdecomposition of the carbohydrate chain may be carried out, for example,by hydrolysis with acids or alkalies or β-glucanase, acetolysis, orsonication. The fractionation based on molecular weight may be carriedout by using a fractional precipitation method with organic solventssuch as alcohols, acetone or ethers or salts or by fractionation with amolecular sieve agent or a molecular sieve membrane.

Part of the carbohydrate chains of the polyglucosides exemplified in (1)to (3) above may be chemically modified with an alkyl group such as amethyl group, a hydroxyalkyl group such as a hydroxymethyl group, acarboxyalkyl group such as a carboxymethyl group, an acid group such asan acetyl group, a sulfuric acid group, a phosphoric acid group oranother functional group. Such a functional group may be introduced bymethods known per se [see, for example, (1) "Seikagaku Kenkyuhou I"(Methods of Studying Biochemistry I") 283-303 (1967) edited by Ando,Terayama, Nishizawa and Yamakawa, Asakura Shoten, and (2) Whistler, R.L. ed.: Methods in Carbohydrate Chemistry III, 193-267, 271-331 (1964),Academic Press, New York and London]. By partial chemical modificationof a (1→3)-β-D-glucan with a molecular weight of at least about 60,000having a factor G activating action, the number of (1→3)-β-D-glucosidestructural units of formula (I) continuously bound to each other in thepoly-(1→3)-β-D-glucoside structure portion is adjusted to 370 or less.This chemically modified glucan may be used as the inhibitor of thisinvention.

Specific examples of the polyglycoside preferably used in this inventionare shown below.

Laminarioligosaccharide having a molecular weight of 342 to 1,638;

laminaridextrin having a molecular weight of 1,800 to 3,258;

(1→3)-β-D-glucan having an average molecular weight of 2,000 to 60,000;

laminaran having an average molecular weight of 3,000 to 23,000;

sclerotan having an average molecular weight of 3,000 to 20,000,

schizophyllan having an average molecular weight of not more than500,000;

lentinan having an average molecular weight of not more than 1,100,000;

a water-soluble function of baker's yeast glucan having an averagemolecular weight of not more than 12,000;

lichenan having an average molecular weight of not more than 33,000;

barley β-glucan having an average molecular weight of not more than200,000;

partially carboxymethylated (1→3)-β-D-glucan having an average molecularweight of 40,000 to 240,000 obtained, for example, by partialcarboxymethylation of Curdlan, and its salts (the degree ofsubstitution: 0.003 to 1.0).

Partially carboxymethylated laminaran having an average molecular weightof not more than 23,000 and its salts (the degree of substitution: notmore than 1.0);

partially methylated (1→3)-β-D-glucan having an average molecular weightof not more than 80,000 (the degree of substitution: 0,003 to 1.0); and

partially sulfated laminaran having an average molecular weight of notmore than 23,000 and its salt (the degree of substitution: not more than1.0).

As will be demonstrated by working examples given later on, thepolyglycosides in accordance with this invention have the action ofstrongly inhibiting the activation of factor G in LAL, and therefore canbe used to specifically detect and assay endotoxins in an assay sampleby the Limulus test without adverse effects by the factor G system. Thepolyglycoside as a factor G activation inhibitor may be used in theLimulus test in an amount larger than that required to inhibit theactivation of factor G in LAL completely. The polyglycoside may be addedeither (1) at the time of detection and assay, (2) to LAL beforehand, or(3) at the time of extracting and preparing LAL.

The amount of the inhibitor required to completely inhibit activation offactor G in LAL can be determined by, for example, the followingprocedure. Under ice cooling, a fixed amount of a factor G activatingsubstance (containing no endotoxin or containing a minimum of a factor Gactivation inhibitor) sufficient to activate LAL under ordinary assayconditions is added to a fixed amount of LAL. To the mixture is added aninhibitor (free from an endotoxin) in varying concentrations. Thereaction is carried out under the same conditions as in the case ofusing ordinary LAL. Under these conditions, the concentration of theinhibitor which inhibits the activation of LAL completely is determined.

The inhibitor having the above-determined concentration is added to afixed amount of LAL, and the factor G activating substance is furtheradded in varying amounts, and it is confirmed that in any concentrationof the factor G activating substance, LAL is not activated.

By the above operation, the amount (concentration) of the inhibitorrequired to completely inhibit the activation of factor G in a fixedamount of LAL can be determined.

The amount of the factor G activation inhibitor required to completelyinhibit the activation of factor G in commercial lysates is shown inTable 1.

                  TABLE 1                                                         ______________________________________                                                                GI*                                                   Lysate (tradename)      (ng/ml LAL)                                           ______________________________________                                        Pregel (Seikagaku Kogyo Co., Ltd.)                                                                    120                                                   Pregel-S (Seikagaku Kogyo Co., Ltd.)                                                                  120                                                   Limulus-Test wako (Wako Pure Chemical                                                                 100                                                   Industries, Ltd.)                                                             Limulus HS-Test wako (Wako Pure Chemical                                                              100                                                   Industries, Ltd.)                                                             Pyrotell (Cape Cod, Inc.)                                                                             120                                                   Pyrosate (Haemachem Inc.)                                                                             230                                                   Pyrotest (Difco Laboratories Inc.)                                                                    230                                                   Pyrogent (Whittaker Bioproducts, Inc.)                                                                 50                                                   Pyrodick (Seikagaku Kogyo Co., Ltd.)                                                                  230                                                   Toxicolor (Seikagaku Kogyo Co., Ltd.)                                                                 450                                                   QCL-1000 (Whittaker Bioproducts, Inc.)                                                                 50                                                   Coatest Endotoxin (Kabi Vitrum)                                                                       230                                                   ______________________________________                                         (Note) *factor G activation inhibitor: GPC fraction 4 of the formic           aciddegraded product of Curdlan obtained in Preparation Example 41 (sampl     No. 14 in Table 2).                                                      

As is clear from the required amounts of the inhibitors, the suitableamount of the polyglycoside in accordance with this invention to becontained in the Limulus test reagent composed of LAL is at least 50 ng,preferably at least 100 ng, more preferably 100 to 230 ng, especiallypreferably 230 to 500 ng, per ml of LAL.

The method of producing the factor G activation inhibitors of thisinvention, their functional mechanism, Limulus test reagent kitscomprising the inhibitors will be described in detail.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a molecular sieve GPC fractionation pattern of commercialCurdlan; and

FIG. 2 shows re-chromatographic fractionation patterns of fractions Nos.44 to 46 in FIG. 1.

METHOD OF PREPARATION FACTOR G ACTIVATION INHIBITOR OF THE INVENTION

The factor G activation inhibitor of this invention can be prepared, forexample, by the methods shown in the following Preparation Examples.Commercial (1→3)-β-D-glucans which are within the scope of thisinvention may be directly used.

Preparation Example 1

Preparation from commercial Curdlan by molecular sieve chromatographicfractionation:

One gram of sample No. 101 Curdlan (a reagent produced by Wako PureChemical Industries, Lot No. PEQ 9080, Mn>136,000, Mw/Mn>2.76) wasdissolved in 0.3M NaOH in a concentration of 5 mg/ml. Each 100microliter aliquot of the solution was subjected to gel permeationchromatography (GPC hereinafter) under the following conditions.

Columns: TSK gel G6000 PW_(XL) and G5000 PW_(XL) (both 7.8×300 mm)connected in series

Mobile phase: 0.3M NaOH

Flow rate: 0.5 ml/min.

Low molecular weight fractions (Nos. 44 to 46) eluted were collected andagain subjected to chromatography to give 0.015 mg of a sample (sampleNo. 1) having a number average molecular weight of 3,050 and apolydispersity of 1.29. The GPC fractionation pattern is shown inFIG. 1. The fractionation patterns of fractions Nos. 44 to 46 in FIG. 1which were again chromatographed are shown in FIG. 2.

Sample No. 1 was digested with β-1,3-glucanase (Zymolyase 100T, aproduct of Seikagaku Kogyo Co., Ltd.). The enzyme digestion product wasanalyzed by GPC under the following conditions.

Columns: TSK gel G4000 PW_(XL), G3000 PW_(XL) and G2500 PW_(XL)connected in series

Mobile phase: Distilled water

Flow rate: 0.6 ml/min.

The carbohydrate composition of the enzyme-digestion product wasdetermined to be: glucose 40%, laminariribose 30%, laminaritriose 20%,laminaritetraose 8%, laminaripentaose 2% (recovery ratio 94%). Theanalysis showed that the carbohydrate structure of sample No. 1 isβ-polyglucoside having a (1→3)-β-D-glucoside structure portion.

Preparation Example 2

Fractionation of Curdlan based on the difference of solubility in water:

Fifty grams of commercial Curdlan (sample No. 101) was suspended indistilled water, and fractionated by operations shown in the followingflowsheet. ##STR8##

Preparation Example 3

Preparation of a water-insoluble carbohydrate fraction of Curdlan bydecomposition with formic acid:

45 g of sample No. 102 was decomposed with formic acid by the method ofK. Ogawa et al. [Carbohydr. Res., 29, 397-403 (1973)). The operationsare shown in the following flowsheet. ##STR9##

Preparation Example 4-1

Re-fractionation by a molecular sieve of a water-soluble fraction of aformic acid decomposition product of Curdlan:

0.15 g of the water-soluble fraction (sample No. 3) obtained inPreparation Example 3 was dissolved in 30 ml of distilled water, and byGPC (column: TSK gel G3000 PW_(XL) ×2, G2500 PW_(XL) ×1; mobile phase:distilled water; flow rate: 0.5 ml/min.), 0.5 ml fractions werecollected. By re-chromatography, six samples (Nos. 11 to 16) havingdifferent molecular weights were obtained.

Preparation Example 4-2

Re-fractionation by a molecular sieve of a water-insoluble fraction of aformic acid degradation product of Curdlan:

0.2 g of the water-insoluble fraction (sample No. 4) obtained inPreparation Example 3 was dissolved in 40 ml of 0.3M NaOH solution, andfractionated and re-chromatographed in the same way as in PreparationExample 4-1 by GPC (column: TSK gel G3000 PW_(XL) ×2, G2500 PW_(XL) ×1;mobile phase: 0.3M NaOH solution; flow rate: 0.5 ml/min). A 0.3M HClsolution was added to the eluates for neutralization, and two samples(Nos. 17 and 18) having different molecular weights were obtained.

Preparation Example 5

Preparation of a sample by sonication of a water-insoluble fraction ofCurdlan:

One gram of sample No. 102 was suspended in about 100 ml of 5 mM NaOH,and under ice cooling, sonicated at 20 kHz and 80 kW for 12 minutes bySonicator® (model 5202 PZT, made by Ohtake Works, Tokyo) to depolymerizeit.

5M NaOH was added to the treated solution to prepare a 0.3M NaOHsolution of the above sample, and fractionated by chromatography as inPreparation Example 4-2 to obtain eight samples (Nos. 19 to 22 and 103to 106) having different molecular weights.

Preparation Example 6-1 Preparation (I) of an inhibitor derived from asea alga:

By the method of T. Usui et al., Agric. Biol. Chem. 43, 603-611 (1979),a commercial dry frond of Eisenia bicyclis (100 g; purchased from SuitaSyoten, Co., Ltd., Tokyo) was ground to a powder and treated with 80%ethanol to remove a low-molecular-weight soluble fraction. A laminaranfraction was extracted from the residue by using a 2% aqueous solutionof CaCl₂. 95% ethanol was added to the extract to prepare a solution ofthe laminaran fraction having a final ethanol concentration of 75%. Theresulting precipitate was collected by centrifugal sedimentation andthen washed with ethanol to obtain a crude laminaran sample. The crudesample was re-dissolved in distilled water and subjected to an anionicexchanger (DEAE-Toyopearl) to remove acidic substances (alginic acid,etc.) and pigments. The residual solution was again precipitated withethanol to give sample No. 25.

Preparation Example 6-2

Preparation (II) of an inhibitor derived from a sea alga:

In accordance with the method of J. J. Connell et al., J. Chem. Soc.,3494 (1950), 100 g of a commercial dry frond of Laminaria japonica(purchased from Suita Syoten Co., Ltd., Tokyo) was ground to a powder,and extracted with 0.09M HCl solution for 3 days. The insoluble matterwas separated by filtration, and the filtrate was left to stand for 1day. A small amount of precipitate that formed was removed bycentrifugal separation, and 3 volumes of ethanol was added to thesupernatant. The ppt was washed with an alcohol and dried to give awater-soluble laminaran fraction (sample No. 27).

Preparation Example 7-1

Preparation (I) of an inhibitor derived from eumycetes:

Scretotan derived from Sclerotinia libertiana was obtained as follows:

By the method of Kitahara et al., Res. Bull. Fac. Agri. Gifu University8, 100-105 (1957), a de-fatted dry powder (30 g) of the sclerotium ofSclerotinia libertiana was extracted fully with water. The residue wasextracted with a 7% aqueous solution of NaOH. 10% CuSO₄ solution wasadded to the extract to form a precipitate. The precipitate wasseparated, washed with hydrochloric acid in methanol to remove copper,washed with 80% methanol to remove HCl and washed with methanol andether, and dried. This procedure was repeated three times to purify thematerial, and 6 g of sample No. 28 was obtained.

Preparation Example 7-2

Preparation (II) of an inhibitor derived from eumycetes:

A sample derived from Schizophyllum commune was obtained as follows:

By the method of Tabata et al., Carbohydr. Res., 89, 121-135 (1981),commercial schizophyllan (tradename Sonifilan produced by Kaken ChemicalCo., Ltd.; medicine Lot No. J61040) was sonicated in aqueous solutionfor 10 hours in accordance with the procedure of Preparation Example 5.By fractionation with a molecular sieve under an alkaline condition,three samples (Nos. 29, 30 and 31) having different molecular weightswere obtained.

Preparation Example 7-3

Preparation (III) of an inhibitor derived from eumycetes:

A β-glucan sample derived from baker's yeast, Saccharomyces cerevisiaewas obtained as follows:

Distilled water (50 ml) was added to 90 mg of commercial bakers' yeastglucan (Lot No. 56F-4027, a product of Sigma Chemical Co.), and themixture was stirred at room temperature for 2 hours, and thencentrifuged. About 50 ml of the supernatant was concentrated to 1 mlunder reduced pressure. The insoluble matter was again removed bycentrifugation, and 0.64 mg of sample No. 33 was obtained from thesupernatant.

Preparation Example 8

Preparation of a sample from barley β-glucan-:

Commercial barley β-glucan (a product of Sigma Chemical Co.; Lot No.56F-0652) was dissolved in 0.3M NaOH to form its solution in aconcentration of 5 mg/ml. By fractionation with a molecular sieve in analkaline condition, a β-glucan sample (No. 36) having a narrow molecularweight distribution was prepared.

Separately, the above barley β-glucan was dissolved in hot water at aconcentration of 5 mg/ml. The solution was centrifuged (3,500 rpm, 10minutes). 100 μl of the supernatant was subjected 50 times to GPC usingdistilled water as a mobile phase in accordance with Preparation Example4-1. The fractions were re-fractionated under the same conditions togive two samples (Nos. 37 and 38) having different molecular weights.

Preparation Example 9

Preparation of partially carboxymethylated (1→3)-β-D-glucan (averagedegree of substitution: 0.63):

A water-insoluble fraction of Curdlan obtained in accordance withPreparation Example 2 was carboxymethylated by the method of A. E.Clarke and B. A. Stone [Phytochemistry 1, 175-188 (1962)]. 100 g of thewater-insoluble fraction of Curdlan was dissolved at 0° C. in 1 liter ofa 5M aqueous solution of sodium hydroxide in a gaseous nitrogen flow.With stirring, a solution of 236 g of monochloroacetic acid in 200 ml ofwater was added dropwise. After the addition, the solution was stirredat 60° to 65° C. for 2 hours. The resulting gel was strongly agitated in2.5 volumes of ethanol to form fine fragments and filtered. The residuewas washed fully with 70% ethanol, then further washed with ethanol andether, and dried. The product was dissolved in 7 liters of water, andneutralized with 1M acetic acid. Activated charcoal (40 g) was added.The mixture was stirred at room temperature for 1 hour, and filtered.The filtrate was concentrated under reduced pressure to a volume of 1liter. Ethanol in three times its volume was added to the concentrate toform a precipitate. The precipitate was washed with ethanol and ether,and dried under reduced pressure on concentrated sulfuric acid to give113.85 g of a partially carboxymethylated (1→3)-β-D-glucan.

The resulting glucan was found to have a degree of etherification (thedegree of substitution) of 0.63 by measurement in accordance with theuranyl nitrate method of D. F. Durso [Methods in Carbohydrate Chem.,VIII 127-129 (1980)]. This means that out of 3 hydroxyl groups in oneglucose residue forming the polysaccharide chain, 0.63 hydroxyl groupswere substituted.

A 25 mg aliquot of the resulting partially carboxymethylated(1→3)-β-D-glucan was dissolved in 5 ml of a 0.1M aqueous solution ofammonium acetate, and fractionated by GPC (column: Toyopearl HW65F,5×100 cm; mobile phase: 0.1M aqueous solution of ammonium acetate; flowrate: 5.8 ml/min), and re-fractionated by GPC using other columns(columns: TSK gel G6000 PW_(XL) +G500 PW_(XL) connected in series;mobile phase: 0.1M aqueous solution of ammonium acetate; flow rate: 0.6ml/min.) to obtain sample No. 41 (Mn=231,000) having a narrow molecularweight distribution.

Furthermore, 0.3 g of the partially carboxymethylated (1→3)-β-D-glucanwas dissolved in 30 ml of distilled water, and sonicated (9 KHz, 180-130W, 1 hour) by a sonicator (Insonator Model 201M, made by Kubota Works)to depolymerize it. A portion (4.5 ml) of the depolymerized product wasmixed with 0.5 ml of a 1M aqueous solution of ammonium acetate. Themixture was subjected to GPC fractionation and GPC re-fractionation bythe same procedure as in the procedure of obtaining sample No. 41 togive two samples (Nos. 39 and 40) having different molecular weights.

Preparation Example 10

Preparation of a partially carboxymethylated (1→3)-β-D-glucan having adegree of substitution of 1.2:

Ten grams of the carboxymethylated (1→3)-β-D-glucan having a degree ofsubstitution of 0.63 obtained in Preparation Example 9 was added to 25ml of 10.5M NaOH in a gaseous nitrogen flow and prepared into a paste.With vigorous stirring, 10 g (12 ml) of an aqueous solution ofmonochloroacetic acid was added. The mixture was warmed to 60° C. andstirred for 4 hours. After cooling, 30 ml of 2M HCl was added, and thesolution was poured into 200 ml of hydrochloric acid in ethanol (40 mlHCl ethanol). The resulting precipitate was collected, washed with 70%ethanol and further with ethanol and ether, and dried under reducedpressure to give sample No. 107.

The degree of substitution of this sample was 1.20, measured by the samemethod as in the case of Preparation Example 9.

Preparation Example 11

Preparation of partially carboxymethylated laminaran:

Partially carboxymethylated laminaran was prepared by treating laminaran(Lot No. 77F-3885 of Sigma Chemical Co.) of Laminaria digitata by themethod of A. E. Clarke and B. A. Stone: Phytochem. 1, 175 (1962) in thesame way as in the method of partial carboxymethylation in PreparationExample 9. Thus, sample No. 42 having a degree of substitution of 0.06was obtained.

Preparation Example 12

Preparation of partially methylated (1→3)-β-D-glucan:

In accordance with the method of M. Samec, Kolloid-Beihefte 51, 369(1940), 3.0 g of a water-insoluble fraction of Curdlan obtained as inPreparation Example 2 was suspended in 80 ml of water, and in a gaseousnitrogen flow, 1.35 ml of a saturated aqueous solution of sodiumhydroxide was added to dissolve the water-insoluble fraction completely.At 4° C., 60 g of dimethylsulfate was gradually added, and in about 1hour, the reaction solution was added dropwise to acetone. The resultingprecipitate was collected, thoroughly washed with acetone, and driedover conc. sulfuric acid under reduced pressure to give 3.13 g (sampleNo. 43; degree of substitution: 0.16).

Preparation Example 13

Preparation of partially sulfated laminaran:

Sulfation of laminaran from Laminaria digitata was carried out inpyridine by the following procedure using a pyridine-sulfur trioxidecomplex (Lot No. PPL 8823 of Wako Pure Chemical Industries).

0.5 g of thoroughly dried laminaran (Sigma Chemical Co.; Lot No.77F-3885) of Laminaria digitata was dissolved in 50 ml of dehydratedpyridine, and 1 g of a pyridine-sulfur trioxide complex was added. Thereaction was carried out at 60° C. for 1 hour. Water (100 ml) was addedto the reaction mixture with cooling. It was then neutralized withsodium hydroxide and dialyzed against water through a dialysis membrane(spectropore; molecular weight 1,000 cut) which had been washed fullywith an alkaline aqueous solution to remove the glucan. The dialyzatewas concentrated and 2 times its volume of acetone was added toprecipitate the carbohydrate component. The carbohydrate component waswashed with acetone, and dried over conc. sulfuric acid under reducedpressure to give 0.38 g (sample No. 44 with a degree of substitution of0.14).

The degrees of substitution of methyl groups and sulfate groups in thepreparations obtained in Preparation Examples 12 to 13 were measured andcalculated in accordance with the method described in (1) Ochiai, Tsudaand Sakamoto: "Yuki Teiryo Bunsekiho (Biryo)" [(Methods of OrganicQuantitative Analysis (Tiny Amounts)], Nanzando (1956); and (2)Whistler, R. L. ed., Method in Carbohydrate Chemistry III, pages229-235, 227-280 (1964), Academic Press.

Commercial Samples

The properties of the following commercial samples were determined. Thesamples were directly subjected to measurements or followingalkali-solubilization and neutralization.

Glucose (JIS special graded reagent; Wako Pure Chemical Industries;sample No. 108)

Laminarioligosaccharides (pure reagent, Seikagaku Kogyo Co., Ltd.;samples Nos. 5 to 10)

Laminaran derived from Eisenia araborea (reagent of Nakarai ChemicalCo., Ltd.; sample No. 23)

Laminaran derived from E. araborea (Tokyo Kasei Kogyo Co., Ltd.; sampleNo. 24)

Laminaran derived from Laminaria digitata (reagent of Sigma ChemicalCo.; sample No. 26)

Lentinan derived from Lentinus edodes (a product of YamanouchiPharmaceutical Co., Ltd., medicine lot No. CKC7; sample No. 32)

Lichenan derived from Cetraria islandica (reagent of Sigma Chemical Co.;sample No. 34)

Lichenan derived from Usnea barbata (reagent of Sigma Chemical Co.;sample No. 35)

EXAMPLES 1-44

The molecular weights and factor G activation inhibiting titers of theabove samples were measured, and the results are shown in Table 2.

                                      TABLE 2                                     __________________________________________________________________________                                                  Factor G                                                       Carbo-         activation                                                     hydrate        inhibiting                      Sample             Method of   structure                                                                          Mn        titer                           No. Substance      preparation (*1) (*2) Mw/Mn                                                                              (units/mg)                      __________________________________________________________________________     1  GPC fraction of Curdlan                                                                      PE (*3) 1   (1)  3,050                                                                              1.29 1,000,000                        2  Water-soluble fraction                                                                       PE 2        (1)  3,270                                                                              2.49 2,240,000                           of Curdlan                                                                    Formic acid degradation                                                       product of Cardlan                                                         3  Water-soluble fraction                                                                       PE 3        (1)  2,080                                                                              1.90 10,700,000                       4  Water-insoluble fraction                                                                     PE 3        (1)  10,000                                                                             3.19 324,000                          5  Laminasiribose Commercial  (1)  342       214                                                (Seikagaku Kogyo)                                           6  Laminaritriose Commercial  (1)  504       4,670                                              (Seikagaku Kogyo)                                           7  Laminaritetraose                                                                             Commercial  (1)  667       20,000                                             (Seikagaku Kogyo)                                           8  Laminaripentaose                                                                             Commercial  (1)  829       39,800                                             (Seikagaku Kogyo)                                           9  Laminarihexaose                                                                              Commercial  (1)  991       55,000                                             (Seikagaku Kogyo)                                          10  Laminariheptaose                                                                             Commercial  (1)  1,153     103,000                                            (Seikagaku Kogyo)                                              Formic acid degradation                                                       product of Curdlan                                                        11  GPC fraction 1 PE 4-1      (1)  2,370                                                                              1.20 708,000                         12  GPC fraction 2 PE 4-1      (1)  3,400                                                                              1.20 13,400,000                      13  GPC fraction 3 PE 4-1      (1)  4,800                                                                              1.20 20,000,000                      14  GPC fraction 4 PE 4-1      (1)  5,800                                                                              1.20 31,600,000                      15  GPC fraction 5 PE 4-1      (1)  6,800                                                                              1.20 6,310,000                       16  GPC fraction 6 PE 4-1      (1)  9,800                                                                              1.22 3,980,000                       17  GPC fraction 7 PE 4-2      (1)  14,500                                                                             1.24 1,820,000                       18  GPC fraction 8 PE 4-2      (1)  27,500                                                                             1.26 126,000                             Sonicated product of Curdlan                                              19  GPC fraction 1 PE 5        (1)  20,700                                                                             1.27 646,000                         20  GPC fraction 2 PE 5        (1)  28,300                                                                             1.18 389,000                         21  GPC fraction 3 PE 5        (1)  50,200                                                                             1.26 4,900                           22  GPC fraction 4 PE 5        (1)  58,100                                                                             1.29 234                                 Laminaran                                                                 23  from Eisenia araborea                                                                        Commercial  (2)a)                                                                              16,800                                                                             1.49 6,760                                              (Nakarai Chemical)                                         24  from E. araborea                                                                             Commercial  (2)a)                                                                              11,200                                                                             1.55 29,500                                             (Tokyo Kasei)                                              25  from E. bicyclis                                                                             PE 6-1      (2)a)                                                                              22,500                                                                             1.27 64,600                          26  from Laminaria digitata                                                                      Commercial  (2)b)                                                                              5,850                                                                              1.16 7,080,000                                          (Sigma Chemical)                                           27  from L. japonica                                                                             PE 6-2      (2)b)                                                                              17,700                                                                             3.98 39,800                          28  Sclerotan      PE 7-1      (2)d)                                                                              16,800                                                                             2.77 26,300                              Schizophyllan                                                             29  GPC fraction 1 PE 7-2      (2)d)                                                                              6,750                                                                              3.14 138,000                         30  GPC fraction 2 PE 7-2      (2)d)                                                                              23,600                                                                             2.37 11,700                          31  GPC fraction 3 PE 7-2      (2)d)                                                                              27,500                                                                             1.49 50,100                          32  Lentinan       Commercial  (2)d)                                                                              21,200                                                                             2.63 10,000                                             (Yamanouchi Pharm.)                                        33  Water-soluble fraction                                                                       PE 7-3      (2)e)                                                                              11,600                                                                             5.14 11,500                              of bakers' yeast glucan                                                       Lichenan                                                                  34  from Cetraria islandica                                                                      Commercial  (3)  22,000                                                                             4.72 3,550                                              (Shigma Chemical)                                          35  from Usnea barbata                                                                           Commercial  (3)  23,200                                                                             4.07 120                                                (Shigma Chemical)                                              Barley β-glucan                                                      36  GPC-fraction 1 PE 8        (3)  54,900                                                                             1.16 30,900                          37  GPC fraction 2 PE 8        (3)  129,000                                                                            1.09 11,700                          38  GPC fraction 3 PE 8        (3)  200,000                                                                            1.13 40,700                              Partially carboxymethylated                                                   (1 → 3)-β-D-glucan                                                (DS = 0.63)                                                               39  GPC fraction 1 PE 9        (1)  42,400                                                                             1.14 117,000                         40  GPC fraction 2 PE 9        (1)  77,300                                                                             1.10 91,200                          41  GPC fraction 3 PE 9        (1)  231,000                                                                            1.10 80,000                          42  Partially carboxymethylated                                                                   PE 11      (2)b)                                                                              8,170                                                                              1.21 3,630,000                           laminaran                                                                 43  Partially methylated                                                                          PE 12      (1)  78,200                                                                             1.10 93,300                              (1 → 3)-β-D-glucan                                            44  Partially sulfated                                                                            PE 13      (2)b)                                                                              10,300                                                                             2.04 117,000                             laminaran                                                                 101 Curdlan        Commercial  (1)  136,000<                                                                           2.76<                                                                              100>                                               (Wako Pure Chemical)                                       102 Water-insoluble fraction                                                                     PE 2        (1)  159,000<                                                                           2.50<                                                                              100>                                of Curdlan                                                                    Sonicated product of                                                          Cardlan                                                                   103 GPC fraction 5 PE 5        (1)  76,300                                                                             1.26 100>                            104 GPC fraction 6 PE 5        (1)  92,600                                                                             1.23 100>                            105 GPC fraction 7 PE 5        (1)  171,000                                                                            1.19 100>                            106 GPC fraction 8 PE 5        (1)  216,000                                                                            1.19 100>                            107 Partially carboxymethylated                                                                   PE 10      (1)  329,000<                                                                           1.27<                                                                              100>                                (1 → 3)-β-D-glucan                                                (DS = 1.20)                                                               108 Glucose        Commercial       180       100>                                               (Wako Pure Chemical)                                       __________________________________________________________________________     Notes to Table 2                                                              (*1): The numbers are classification numbers given in the specification.      (*2): The molecular weights of glucose and laminarioligosaccharide are        absolute molecular weights (theoretical values), and the molecular weight     of the other samples are those calculated for polyethylene oxide and          polyethylene glycol determined by the measuring method described below.       (*3): PE stands for Preparation Example.                                      Samples Nos. 1 to 44 are factor G inhibitors of this invention, while         samples Nos. 101 to 108 are for comparison.                              

The molecular weights in the table are number average molecular weights(Mn) defined by the following equation and determined by GPC. Themolecular weight distribution is expressed by a polydispersity (Mw/Mn)##EQU1##

In the above equations, Hi represents the height of the ith peak (sampleconcentration) when the chromatogram is multidivided into equal parts bytime, and Mi represents the molecular weight of the ith peak.

The factor G activation inhibiting titer was measured by the "method ofmeasuring the titer of the activity of a factor G activation inhibitor"described below and expressed in units/mg.

Method of measuring the titer of the activity of a factor G activationinhibitor (sometimes abbreviated as GI):

Two hundred microliters of the reaction mixture contained the followingmaterials.

    ______________________________________                                        (1) Assay sample (note 1) GI sample or distilled water                        50 microliters                                                                [factor G activating substance (abbreviated as                                GA, note 2) 10 pg or not added                                                (2) proclotting enzyme fraction of LAL (A.sub.280 = 2.5; note 3)              30 microliters                                                                (3) factor G fraction of LAL (A.sub.280 = 0.9; note 3) 20 microliters         (4) Tris-HCl buffer (pH 8.0) 20 μmoles                                     (5) MgCl.sub.2 20 μmoles                                                   (6) Boc--Leu--Gly--Arg--pNA 0.13 μmole                                     ______________________________________                                    

The above reaction mixture was incubated at 37° C. for 30 minutes, and0.5 ml each of 0.04% sodium nitrite (0.48M HCl solution), 0.3% ammoniumsulfamate, and 0.07% N-(1-naphthyl)ethylenediamine dihydrochloride weresuccessively added, and its color was changed by diazo coupling. Theamount of pNA liberated was measured as the absorbance at 545 nm (A₅₄₅).

The GI activity was calculated from the following equation. ##EQU2##

The amount of GI which inhibited activation of factor G by GA underthese conditions to an extent of 100% is defined as 100 units.

(Note 1)

A sample which is water-insoluble is used after it is dissolved in 0.3MNaOH, and neutralized with an equal volume of 0.3M HCl.

(Note 2)

A GPC fractionated pure sample of the sonicated product of Curdlan whichwas prepared in Preparation Example 5 (Table 2, sample No. 106 having amolecular weight of 216,000).

(Note 3)

Prepared from horseshoe crab, T. tridentatus, inhabiting the coast ofJapan in accordance with the method described in T. Obayashi et al.,Clin. Chim. Acta, 149, 55-65 (1985).

The following conclusions may be drawn from the data given in Table 2.

(a) Commercial Curdlan (sample No. 101) known as a factor G activatingsubstance contains a factor G activation inhibitor which iswater-soluble and has a low molecular weight (samples Nos. 1 and 2).

(b) polyglycosides in which the number of connected (1→3)-β-D-glucosidestructural units constituting the (1→3)-β-D-glucoside structure portionis 2 to 370 show a factor G activation inhibiting action.

(c) Fractions having a molecular weight of not more than 60,000 obtainedby various depolymerization operations from the high molecular weightβ-glucan fraction (sample No. 102) with no appreciable inhibitoryactivity exhibited factor G activation inhibiting titers (samples Nos.3, 4 and 11 to 22).

(d) Fractions (Preparation Example 8, Samples Nos. 36, 37 and 38)obtained from barley glucan in which poly(1→3)-β-D-glucoside structureportions having a degree of not more than 10 andpoly-(1→4)-β-D-glucoside structural portions are linked to each other inblocks [see Ballance et al., Carbohyd. Res., 61, 107-118 (1978)] showactivity equivalent to laminaritetraose or laminaripentaose, and can beused as the inhibitor of the invention whether the glucan as a whole hasa molecular weight of more than 60,000 or less than 60,000.

(e) The partially carboxymethylated (1→3)-β-D-glucan having an averagedegree of substitution of at least 1.0 obtained in Preparation Example10 (sample No. 107 (DS=1.2) lost its factor G activation inhibitingeffect. When sample No. 26 having a high factor G activation inhibitingtiter was partially carboxymethylated (Preparation Example 11) to reducethe chain length of the (1→3)-β-D-glucoside portion (sample No. 42), andwhen in Preparation Example 13, the (1→3)-β-D-glucoside structureportion was shortened by sulfate modification, the factor G activationinhibiting effects were reduced.

(f) When glucan having a degree of polymerization of at least 370 in the(1→3)-β-D-glucoside structure portion and a molecular weight of morethan 60,000 was partially methylated (preparation Example 12) orpartially carboxymethylated (Preparation Example 9, sample No. 41) toattain the structure defined in this invention, the resulting producthad a factor G activation inhibitory effect.

Operating mechanism of the inhibitor of this invention on factor G

The factor G activation pathway in the horseshoe crab blood coagulatingsystem is shown by the following chart, as reported in T. Morita et al.,FEBS Letters, 129, 318-321 (1981). ##STR10##

The following experiment was conducted in order to determine what partof the above coagulation pathway the inhibitor of this inventioninhibits.

A reaction mixture of the following composition was used. Two hundredmicroliters of the reaction mixture contained 5 micrograms of a factor Gactivation inhibitor (laminariheptaose; sample No. 10; abbreviated asI), 3 pg of a factor G activating substance (GPC fraction of sonicatedCurdlan; sample No. 106; abbreviated as A), 20 microliters of a factor Gfraction (abbreviated as G) and 30 microliters of a proclotting enzymefraction (abbreviated as P) prepared from LAL by the method described inT. Obayashi et al., Clin. Chim. Acta, 149, 55-65 (1985), 20 μmoles ofTris-HCl buffer (pH 8.0), 20 μmoles of MgCl₂ and 0.13 μmole of achromogenic substrate Boc-Leu-Gly-Arg-pNA (abbreviated as S).

In each of Runs 1 to 5, the sequence of adding the ingredients and theincubating conditions were varied, and the degree of inhibition of thefactor G pathway was measured and determined in comparison with acontrol in which no inhibitor was added. The results are shown below.

    __________________________________________________________________________                                             Inhibition                                                                    rate (%) to                          Experiment                               the control                          __________________________________________________________________________     ##STR11##                               100                                   ##STR12##                               100                                   ##STR13##                               100                                   ##STR14##                               1.7                                   ##STR15##                               0                                    __________________________________________________________________________

It is clear from Runs Nos. 1 to 3 that when the inhibitor (I) of thisinvention was added to factor G (precursor), the activation of factor Gwas inhibited to an extent of 100% irrespective of the presence of thefactor G activating substance (A).

On the other hand, it is clearly seen from Runs Nos. 4 and 5 that oncefactor G was activated by (A), the activated factor G did not undergoinhibition even in the presence of the inhibitor (I).

It can be concluded therefore that the inhibitor (I) of this inventionacts only on factor G (precursor).

In the presence of the inhibitor (I) of this invention in an amountsufficient to inhibit 100% of factor G in LAL, factor G does not undergoactivation in whatever large amount of the activating substance (A) ispresent. It was confirmed however that if a large amount of theactivating substance (A) is present in the presence of the inhibitor (I)in an amount smaller than that required to inhibit factor G to an extentof 100%, that portion of factor G which remains uninhibited by theinhibitor (I) is activated by the activating substance (A).

Accordingly, the existence of the maximum activating concentration ofthe (1→3)-β-D-glucan derivatives pointed out by A. Kakinuma et al.,Biochem. Biophys. Res. Commun., 101, 434-439 (1981) or the variousβ-glucans shown by T. Morita et al., Prog. Chim. Biol. Res., 189, 53-64(1985) in their ability to activate horseshoe crab factor G could beanalyzed by the above experiment and analysis.

EXAMPLE 45

Comparison of the ability with specific assay of endotoxins between akit containing a factor G activation inhibitor and a kit not containingit:

By using various assay samples, endotoxin specificities were compared bythe following procedure when the factor G activation inhibitor of theinvention was added to LAL-Test and when it was not added to LAL-Test.

The LAL-Test was a Toxicolor Test (colorimetry, Seikagaku Kogyo Co..,Ltd.) having the following composition.

(1) Perchloric acid, (2) sodium hydroxide, (3) buffer, (4)lysate+chromogenic substrate, (5) endotoxin-free distilled water, (6)standard endotoxin, (7) hydrochloric acid, (8) sodium nitrite, (9)ammonium sulfamate, (10) N-(1-naphthyl)ethylenediamine dihydrochloride.

A group of reaction solutions (kit-A) were prepared by dissolving sampleNo. 13 as a factor G activation inhibitor in the buffer (3) at aconcentration of 5 micrograms/ml, and dissolving the LAL reactionreagent in the solution. A group of reaction solutions (kit-B) wereprepared by dissolving (4) in the buffer (3) not containing theinhibitor. The reactivities of the kits A and B with the assay sampleswere compared and are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Comparison of endotoxin-specificities between                                 the presence and absence of factor G activation inhibitor                                Amount per  Reactivity                                             Sample     0.1 ml of   (ΔA.sub.545 /30 min.)                            No.        the sample  Kit-A      Kit-B                                       ______________________________________                                        1. Endotoxin                                                                             2.5 pg      0.447      0.448                                       (E.T.) (*1)                                                                              5.0 pg      0.895      0.894                                       2. Factor G activ-                                                                       3.0 pg      0.000      0.232                                       ating substance                                                                          100 ng      0.001      1.5<                                        (*2)                                                                          3. Factor G activ-                                                                       3.0 pg + 2.5 pg                                                                           0.447      0.678                                       ating substance                                                               + E.T.                                                                        4. Washing from                                                                          8.0 pg      0.001      0.312                                       dialysis                                                                      membrane                                                                      (*3)                                                                          5. Washing from                                                                          8.0 pg + 2.5 pg                                                                           0.446      0.757                                       dialysis                                                                      membrane                                                                      + E.T.                                                                        6. Clinical                                                                              as                                                                 samples    plasma                                                              a(*4)     0.017 ml    0.003 + 0.002                                                                            0.008 + 0.004                                b         "           0.265      0.272                                        c         "           0.261      0.266                                        d         "           0.213      0.210                                        e         "           0.131      0.138                                        f         "           0.101      0.107                                        g         "           0.073      0.071                                        h         "           0.000      1.166                                        i         "           0.000      0.904                                        i         "           0.003      0.304                                        k         "           0.001      0.203                                        l         "           0.004      0.152                                        m         "           0.007      1.5<                                         n         "           0.006      1.5<                                         o         "           0.006      1.5<                                         p         "           0.003      1.5<                                         q         "           0.005      1.5<                                         r         "           0.004      1.5<                                        ______________________________________                                         Notes to Table 3:                                                             (*1): Escherichia coli 0111:B4 derived endotoxin (Difco Laboratories Inc.     (*2): Waterinsoluble fraction of Curdlan; molecular weight >159,000;          sample No. 102 in Table 2.                                                    (*3): Washing resulting from perfusion of distilled water through a hollo     fibertype hemodialyzer (AMNeo-3000; a product of Asahi Medical Co., Ltd.)     constructed by using a cuprammonium rayon membrane (regenerated cellulose     membrane). Its carbohydrate content was determined by the phenolsulfuric      acid method.                                                                  (*4): Values for normal samples (n = 25) are means ± standard              deviations.                                                              

Samples b to j were suspected of having a complication of sepsis. Bloodcultures were positive for Escherichia coli in samples b to e; forPseudomonas aeruginosa in samples f and g; for Candida albicans insample i; and for Candida guilliermondii in sample j. Sample h was froma case of pulmonary aspergillosis, samples k and l from cases ofsystemic fungal infection determined at autopsy, and samples m to r fromcases of chronic renal failure (without microbial infection) underhemodialysis with a hollow fiber-type hemodialyzer made by usingcuprammonium rayon (regenerated cellulose).

Samples Nos. 1 to 5 were directly dissolved in a solvent, and 0.1 ml ofeach solution was used in the reaction, in accordance with the manual ofthe Toxicolor Test. Samples No. 6 (a to r) were obtained by pretreatingplasma samples with the components (1) and (2) of the kit, and 0.1 ml ofeach of the pretreated products was used in the reaction, in accordancewith the method of T. Obayashi [J. Lab. Clin. Med., 104, 321-330(1984)].

Each of the samples was added to the reaction solutions prepared byusing components (3) and (4), and reacted at 37° C. for 30 minutes. Theresulting pNA was colored with coupling reagents (7) to (10). Thereactivities of kits A and B were expressed by absorbance at 545 nm.

The maximum reactivity of the present kit composition , ΔA₅₄₅, was 1.5.

As can be seen from Table 3, kit-A containing the factor G activationinhibitor of the invention and kit-B as a conventional kit notcontaining the inhibitor showed the same reactivity with the endotoxin(sample No. 1). Kit-B showed a very high level of reactivity with thewater-insoluble fraction of Curdlan (sample No. 2) as the factor Gactivating substance. On the other hand, kit-A showed no reactivity withthe above sample, but when the endotoxin (sample No. 1) was used incombination (sample No. 3), kit-A showed the same reactivity as withendotoxin (sample No. 1).

When a washing obtained by washing a cellulose dialysis membrane, knownas a Limulus test-reactive material (non-endotoxic), was used as asample (Nos. 4 and 5) [F. C. Pearson et al., Artif. Organs, 8, 291-298(1984)], both kit-A and kit-B showed the same behaviors and results asin the case of water-insoluble fraction of Curdlan and/or the absence ofan endotoxin.

The foregoing results led to the finding that the combined use of LALwith the factor G activation inhibitor of this invention makes possiblethe specific determination of an endotoxin.

The following can be said with respect to clinical blood samples forwhich the conventional LAL-Test cannot clearly determine whether thepatient suffered from a case of true endotoxemia. Both kits-A and -Bshowed high reactivity with samples No. 6, b to q in which the presenceof Gram-negative bacteria was determined by culturing. On the otherhand, kit-A showed no reactivity, and kit-B showed high reactivity, withsamples (No. 6, h to l) in which the presence of eumycetes known to have(1→3)-β-D-glucan on its cellular wall was determined. Further, kit Ashowed no reactivity with samples (No. 6; m to r) of cases of chronicrenal failure under hemodialysis which cannot be regarded as sufferingfrom endotoxemia in view of the clinical symptoms. Abnormally highvalues obtained with kit-B is presumably because of (1→3)-β-D-glucanderived from the dialysis membrane.

Assaying of clinical samples suspected of infections and sepsis in whichthe presence of an endotoxin is not clear, like the samples describedabove offers the advantage that a true Gram-negative bacterial infection(endotoxemia) can be determined accurately, and that it can also detectmycosis. Hence, it permits early diagnosis of invading microbes, andenables proper selection of medication and treatment and also ananalysis of a therapeutic effect. The provision of a kit containing theinhibitor of this invention can be expected to contribute greatly to anadvance in medicine, particularly diagnosis and therapy.

The following Referential Examples show the preparation of kits forspecific detection of endotoxins by combining LAL with the factor Gactivation inhibitor of the invention.

Referential Example 1

Method of making an endotoxin-specific assay by adding a factor Gactivation inhibitor to a commercial or a conventional Limulus testreagent at the time of detecting the endotoxin:

1-1. The Limulus test reagent (lyophilized product) was dissolved in acustomary manner in a designated dissolving liquid (distilled water orbuffer), and the factor G activation inhibitor was added together with,or separately from, an assay sample (the sequence of addition isarbitrary).

For example, 0.1 ml of distilled water was added to "Pregel-S"(lyophilized product; Limulus test product by the gellation method;Seikagaku Kogyo Co., Ltd.), and 0.01 ml (120 micrograms/ml LAL) of anaqueous solution of the inhibitor (a GPC fraction 4 of the formic aciddegradation product of Curdlan; No. 14 in Table 2) and 0.1 ml of thesample were added. The mixture was gently shaken, and heated at 37° C.for 60 minutes while it was left to stand. The mixture reacted only withthe endotoxin to form a gel.

1-2. An inhibitor was dissolved in advance in a dissolving liquid forthe Limulus test reagent, and dissolving the Limulus test reagent in theresulting solution. When "Coatest® Endotoxin" (chromogenic Limulus testproduct by Kabi Vitrum) was used, one vial of LAL (lyophilized product)was first dissolved in 1.4 ml of distilled water having dissolvedtherein 0.7 microgram of the inhibitor (laminaran; No. 26 in Table 2). Asample (0.1 ml) was added to 0.1 ml (500 ng/ml of LAL) of the resultingsolution. The mixture was heated at 37° C. for 10 minutes. When 0.2 mlof a buffer containing a synthetic substrate (S-2423) was added and thesolution was heated at 37° C. for 3 minutes, the mixture reacted onlywith the endotoxin and the solution became yellow. In determination, 200microliters of a 50 % acetic acid solution was added, and the absorbanceof the solution at 405 nm was measured.

Referential Example 2

Method of making an endotoxin-specific Limulus test reagent by adding afactor G activation inhibitor to LAL in advance:

2-1. Method of adding the inhibitor to commercial LAL (the so-calledLimulus gellation test reagent) in advance.

When turbidimetric kinetic assay was to be performed by using LimulusHS-Test Wako, a lyophilized product of Wako Pure Chemical Industries,Ltd., one vial of LAL (lyophilized product) was dissolved in 5 ml ofdistilled water having dissolved therein 0.5 microgram of the inhibitor(a GPC fraction 4 of the formic acid degradation product of Curdlan; No.14 in Table 2). 0.1 ml (100 ng/ml of LAL) was put in a reaction testtube, and 0.1 ml of a sample was further added. The mixture was gentlyshaken. The mixture was set at a predetermined light measuring positionof an analysis module (37° C.) of an instrument for turbidimetrickinetic assay (Toxinometer ET-201 supplied by Wako Pure ChemicalIndustries, Ltd.), and a start switch was depressed. The mixture reactedonly with the endotoxin, and the gellation time was displayed.

2-2. Method of achieving the object by adding the inhibitor to LALbefore addition of an assay sample.

For example, when turbidimetry was to be performed by using LAL, 0.01 ml(50 ng/ml of LAL) of a 1M Tris-HCl-1M MgCl₂ buffer (pH 8.0) havingdissolved therein 0.5 microgram of the inhibitor (laminaran; No. 26 inTable 2) per ml was added to 0.1 ml of a lysate extracted from theamebocytes of Tachypleus tridentatus by using a hypotonic buffersolution. Then, 0.1 ml of an assay sample was added to the solution, andthe mixture was heated at 37° C. The mixture reacted only with theendotoxin, and the solution became whitely turbid. The amount of theendotoxin could be determined by measuring the absorbance of thesolution periodically at 660 nm.

Referential Example 3

Method of producing a lysate as a material for an endotoxin-specificLimulus test reagent by adding the factor G activation inhibitor at thetime of extracting and preparing LAL:

Hemolymph was drawn from horseshoe crab (any of Tachypleus tridentatus,T. gigas, Limulus polyphemus, and Carcinoscorpius rotundicauda, andcentrifuged to obtain amebocytes (about 20 g). Then 100 ml of aninhibitor solution obtained by dissolving 2.0 mg/liter of the inhibitor(partially carboxymethylated laminaran; No. 42 in Table 2) in distilledwater or a hypotonic buffer solution such as 0.02M Tris-HCl buffer (pH8.0) was added to the amebocytes. The mixture was homogenized by aWaring blender, and then centrifuged (8,000 rpm; 30 minutes; 4° C.) toseparate it into a supernatant and a precipitate. This operation wasrepeated to give about 150 ml of the supernatants as LAL. A fixed amountof the resulting LAL was used in place of LAL extracted by aconventional method, and Limulus test reagents (gellation method,turbidimetric method, turbidimetric kinetic method, chromogenicsynthetic substrate method) were prepared. The resulting reagent was anendotoxin-specific Limulus test reagent which specifically react onlywith endotoxins.

For production of an endotoxin-specific Limulus test reagent by thechromogenic substrate method, the use of the substrates disclosed, forexample, in U.S. Pat. No. 4,495,294 (entitled: Method for DeterminingBacterial Endotoxin and Kit Therefor) and such substrates asR-Ile-Glu-Ala-Arg-pNA andmethoxycarbonyl-D-hexahydrotyrosyl-Gly-Arg-pNA.AcOH (in which Rrepresents an acetyl group, an α-N-benzoyl group, an α-N-carbobenzoxygroup, an N-tert-butoxycarbonyl group, a p-toluenesulfonyl group orother amino acid N-terminal protecting groups will permit production ofa reagent of high sensitivity.

For example, such a reagent can be produced by adding 1.5 micrograms ofMgCl₂ and 4.0 micrograms of a synthetic substrate(N-tert-butoxycarbonyl-Leu-Gly-Arg-p-nitroanilide) to 0.04 ml of thelysate and lyophilizing the mixture. When 0.1 ml of 0.2M Tris-HCl buffer(pH 8.0) and 0.1 ml of an assay sample were added to the lyophilizedproduct and the mixture was heated at 37° C. for 30 minutes, the mixturereacted only with endotoxin, and the solution assumed yellow.

An LAL reagent for the gellation method, the turbidimetric method andturbimetric kinetic assay could be produced by adding 10.0 micrograms ofMgCl₂ to 0.1 ml of the lysate and lyophilizing the mixture. When thisreagent was used as in Referential Examples 1-1, 2-1 and 2-2, it reactedonly with an endotoxin.

Industrial Utilizability

The factor G activation inhibitor of this invention provides anendotoxin-specific LAL-Test reagent by combining it with LAL and isuseful in assaying clinical samples suspected of infections and sepsisin which the presence or absence of an endotoxin is uncertain. It has anadvantage of being able to determine accurately a true Gram-negativebacterial infection (endotoxemia), and by using a conventional LAL-Testin combination, mycosis can be detected. It permits early diagnosis ofinvading microbes, and enables proper selection of medication andtreatment and also an analysis of a therapeutic effect. The provision ofa kit containing the inhibitor of this invention can be expected tocontribute greatly to an advance in medicine, particularly in diagnosisand therapy.

We claim:
 1. An endotoxin-specific Limulus test reagent consistingessentially of horseshoe crab amebocyte lysate containing factor G and apolyglycoside composed of a poly-(1→3)-β-D-glucoside structural portionconsisting of 2 to 370 (1→3)-β-D-glucoside structural units of thefollowing formula: ##STR16## which are continuously bound to one anotherin an amount of 50 to 500 ng per ml of the horseshoe crab amebocytelysate and sufficient to inhibit 100% of the activation of factor G, andcontaining no activated factor G.
 2. The reagent of claim 1 in which thepoly-(1→3)-β-D-glucoside structural portion consists of 3 to 310(1→3)-β-D-glucoside structural units of formula (I) bound to oneanother.
 3. The reagent of claim 1 in which the polyglycoside islaminarioligosaccharide having a molecular weight of 342 to 1,638. 4.The reagent of claim 1 in which the polyglycoside is laminaridextrinhaving a molecular weight of 1,800 to 3,258.
 5. The reagent of claim 1in which the polyglycoside is a (1→3)-β-D glucan having an averagemolecular weight of 2,000 to 60,000.
 6. The reagent of claim 1 in whichthe polyglycoside is laminaran having an average molecular weight of3,000 to 23,000.
 7. The reagent of claim 1 in which the polyglycoside issclerotan having an average molecular weight of 3,000 to 20,000.
 8. Thereagent of claim 1 in which the polyglycoside is schizophyllan having anaverage molecular weight of not more than 500,000.
 9. The reagent ofclaim 1 in which the polyglycoside is lentinan having an averagemolecular weight of not more than 1,100,000.
 10. The reagent of claim 1in which the polyglycoside is a water-soluble fraction of bakers' yeastglucan having an average molecular weight of not more than 12,000. 11.The reagent of claim 1 in which the polyglycoside is lichenin having anaverage molecular weight of not more than 33,000.
 12. The reagent ofclaim 1 in which the polyglycoside is barley β-glucan having an averagemolecular weight of not more than 200,000.
 13. The reagent of claim 1 inwhich the polyglycoside is partially carboxymethylated (1→3)-β-D glucanor its salt (degree of substitution 0.003 to 1.0) having an averagemolecular weight of 40,000 to 240,000.
 14. The reagent of claim 1 inwhich the polyglycoside is a partially carboxymethylated laminaran orits salt (degree of substitution not more than 1.0) having an averagemolecular weight of not more than 23,000.
 15. The reagent of claim 1 inwhich the polyglycoside is partially methylated (1→3)-β-D glucan (degreeof substitution 0.003 to 1.0) having an average molecular weight of notmore than 80,000.
 16. The reagent of claim 1 in which the polyglycosideis partially sulfated laminaran or its salt (degree of substitution notmore than 1.0) having an average molecular weight of not more than23,000.
 17. The reagent of claim 1 in which the poly-(1→3)-β-D-glucosidestructural portion comprises at least 20% by weight of the reagent. 18.An endotoxin-specific Limulus test reagent consisting essentially ofhorseshoe crab amebocyte lysate containing factor G and a polyglycosidecomposed of a poly-(1→3)-β-D-glucoside structural portion consisting of4 to 180 (1→3)-β-D-glucoside structural units of the following formula:##STR17## which are continuously bound to one another in an amount of 50to 500 ng per ml of horseshoe crab amebocyte lysate and sufficient toinhibit 100% of the activation of factor G, and containing no activatedfactor G.
 19. An endotoxin-specific Limulus test reagent which consistsessentially of horseshoe crab amebocyte lysate containing factor G and apolyglycoside composed of a poly-(1→3)-β-D-glucoside structural portionconsisting of 4 to 180 (1→3)-β-D-glucoside structural units bound to oneanother, having factor G activation inhibiting titer of at least about10,700,000 units/mg and having a molecular weight of about 2,080 toabout 5,800 in an amount of 50 to 500 ng per ml of horseshoe crabamebocyte lysate and sufficient to inhibit 100% of the activation offactor G, and containing no activated factor G.
 20. Anendotoxin-specific Limulus test reagent which consists essentially ofhorseshoe crab amebocyte lysate consisting factor G and a polyglycosidecomposed of a poly-(1→3)-β-D-glucoside structural portion consisting of4 to 180 (1→3)-β-D-glucoside structural units bound to one another,wherein the polyglycoside is a formic acid degradation product ofcurdlan, and which has factor G activation inhibiting titer of at leastabout 10,700,000 units/mg in an amount of 50 to 500 per ml of horseshoecrab amebocyte lysate and sufficient to inhibit 100% of the activationof factor G, and containing no activated factor G.
 21. Anendotoxin-specific Limulus test reagent which consists essentially ofhorseshoe crab amebocyte lysate containing factor G and a polyglycosidecomposed of a poly-(1→3)-β-D-glucoside structural portion consisting of4 to 180 (1→3)-β-D-glucoside structural units bound to one another,wherein the polyglycoside is a formic acid degradation product ofcurdlan, and which has a molecular weight of about 2,080 to about 5,800in an amount of 50 to 500 ng per ml of horseshoe crab amebocyte lysateand sufficient to inhibit 100% of the activation of factor G, andcontaining no activated factor G.
 22. An endotoxin-specific Limulus testreagent which consists essentially of horseshoe crab amebocyte lysatecontaining factor G and a polyglycoside composed of apoly-(1→3)-β-D-glucoside structural portion consisting of 4 to 180(1→3)-β-D-glucoside structural units bound to one another, which has amolecular weight of about 2,000 to 5,800, wherein the polyglycoside is aformic acid degradation product of curdlan, and which has factor Gactivation inhibiting titer of at least about 10,700,000 units/mg in anamount of 50 to 500 ng per ml of horseshoe crab amebocyte lysate andsufficient to inhibit 100% of the activation of factor G, and containingno activated factor G.
 23. The endotoxin-specific Limulus test reagentof claim 19, 20, or 22, in which the polyglycoside has a molecularweight of approximately 2,080 and factor G activation inhibiting titerof approximately 10,700,000 units/mg.
 24. The endotoxin-specific Limulustest reagent of claim 19, 20, 21 or 22, in which the polyglycoside has amolecular weight of approximately 3,400 and factor G activationinhibiting titer of approximately 13,400,000 units/mg.
 25. Theendotoxin-specific Limulus test reagent of claim 19, 20, 21 or 22, inwhich the polyglycoside has a molecular weight of approximately 4,800and factor G activation inhibiting titer of approximately 20,000,000units/mg.
 26. The endotoxin-specific Limulus test reagent of claim 19,20, 21 or 22 in which the polyglycoside has a molecular weight ofapproximately 5,800 and factor G activation inhibiting titer ofapproximately 31,600,000 units/mg.
 27. The endotoxin-specific Limulustest reagent of claim 19, 20, or 22 having factor G activationinhibiting titer of about 10,700,000 to about 31,600,000 units/mg. 28.The reagent of claim 9, 20 or 22 wherein the factor G activationinhibiting titer is measured by adding a factor G activating substance,a proclotting enzyme fraction of horseshoe crab amebocyte lysate, afactor G fraction of horseshoe crab amebocyte lysate, a buffer,magnesium chloride and a chromogenic substrate to a factor G activationinhibitor sample and quantifying an amount of a chromogenic groupliberated after a definite time of incubation.